Compositions for treatment of antibiotic-resistant gram-positive bacterial infections and methods for using and preparing the same

ABSTRACT

Compositions for treatment of antibiotic-resistant gram-positive bacterial infections, as well as methods for using and preparing the same, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional of U.S. application Ser. No. 09/138,127, filed Aug.24, 1998, U.S. Pat. No. 6,025,400.

FIELD OF THE INVENTION

The present invention relates to compositions for treatment ofantibiotic-resistant gram-positive bacterial infections, as well asmethods for using and preparing the same.

BACKGROUND OF THE INVENTION

I. Antibiotic Use and Antibiotic-Resistance

The term “antibiotic” is broadly defined as a chemical compound producedby one microorganism that inhibits the growth of a differentmicroorganism. Today, there are more than 150 antibiotics which areclassified by their chemical structures and mechanisms of action,examples of which are listed in Table 1 below (Lawson et al, TheAmerican Biology Teacher, 6:412-417 (1998)).

TABLE 1 Chemical Class and Mechanism of Action of Common AntibioticsAntibiotic Class Mechanism of Action Ampicillin Broad-spectrum Inhibitscell wall penicillin synthesis Ceftriaxone 3rd generation Inhibits cellwall cephalosporin synthesis Erythromycin Macrolide Inhibit proteinsynthesis Norfloxacin Quinolone Inhibits DNA synthesis StreptomycinAminoglycoside Inhibits protein synthesis Sulfisoxazole SulfonamideCompetitive enzyme inhibitor Tetracycline Tetracycline Inhibits proteinsynthesis

When the first therapeutic antibiotic, penicillin, was introduced in theearly 1940's, many believed that the threat from infectious diseases wasover. However, in the past 25 years, through the abuse and misuse ofantibiotics, many bacteria have developed resistance to theseantibiotics. Today, there are strains of virtually every major bacterialhuman pathogen that are resistant to some of the most effectiveantibiotics. These strains include pathogens that can cause diarrhea,urinary tract infections, otitis media, meningitis, tuberculosis,gonorrhea, pneumonia, dysentery, wound infections, septicemia,bacteremia and surgical infections (Lippe, Breakout: The Evolving Threatof Drug-Resistant Diseases, Sierra Clubs, San Francisco (1995)).

Thus, while 90% of bacterial infections are successfully treated withfirst line antibiotics, there are increasingly situations in which over40% of the infections are resistant to one or more antibiotics(including second line products). The newest antibiotic, vancomycin, hasbeen shown to be the only antibiotic that is effective against somepathogenic bacteria. It has become the last line of defense against someinfections, particularly those by methicillin-resistant Staphylococcusaureus.

In addition, some less pathogenic strains of the genus Enterococcus havevancomycin-resistance genes, and have been shown, in the laboratory, totransfer this resistance to Staphylococcus strains (Lawson et al,supra). As a result, the physician will have no treatment for infectionsby these Staphylococcus strains.

The mechanisms of bacterial resistance to antibiotics include thefollowing:

(1) Loss of cell permeability to the antibiotic;

(2) Enzymes that render the antibiotic ineffective;

(3) Export of the antibiotic out of the cell once it enters the cell;

(4) Modification of the target of the antibiotic; and

(5) Modification of metabolic pathways which result in by-passing thereaction inhibited by the antibiotic.

The seriousness of these mechanisms of antibiotic resistance isaccentuated by the ability of the bacteria to transfer the resistance toother microorganisms, some of which may be fairly genetically unrelatedto the antibiotic-resistant strain. The antibiotic-resistance transfercan occur through one of the following mechanisms: conjugation,transduction, transformation or transposition.

Antibiotic-resistant microorganisms add an estimated $200 million/yearto medical bills. When costs for extended hospital stays are considered,the estimated costs increase by $30 billion/year (Phelps, Medical Care,27:194-203 (1989)). Thus, there is a crucial need for novelantimicrobial agents which can effectively inhibit the growth ofbacteria by mechanisms different from those of existing antibiotics.

II. Traditional Chinese Medical Herbal Formulations

The historic milestones in Traditional Chinese Medicine (TCM) are asfollows:

3494 B.C.: The initial discovery of herbal medicine by emperor ShenNong;

500 B.C.: The flowering of Chinese medicine: medicine, religion, ethics,philosophy; and

16th Century: Li Shizhen (1517-1593) writes Outlines and Divisions ofHerbal Medicines.

The ancient Chinese understood the value of a combinatorial approach tothe treatment of diseases. They believed that a disease can, and often,affects more than a single function and thus, treatment must be directedto multiple targets. A mixture of different herbs was thus, designed toneutralize the multiple effects of a disease. The Western “silverbullet” approach of one single chemical to cure one disease in allpatients has not been readily accepted in TCM.

Most formulations of TCM contain 6 to 12 herbs. Throughout the historyof TCM, there have been many different methods of classifying the waysin which medicinal substances can be combined (Liao Zhong-Chun,Annotated Divine Husbandman's Classic of Materia Medica (1625); andBensky et al, Chinese Herbal Medicine, Materia Medica, Eastland Press,Seattle, Wash. (1993)). Formulations have been carefully crafted so asto accentuate and enhance functionality, to suppress and counteracttoxicity, and to avoid antagonism and incompatibility. Since there areso many possible combinations from such a large collection of herbs, ahierarchical scheme has been developed. That is, the principalingredient is a substance that provides the main therapeutic thrust, thesecond principal ingredient enhances or assists the therapeutic actionsof the first, and the rest of the ingredients serve one or more of thefollowing functions: treatment of accompanying symptoms, moderation ofthe harshness or toxicity of the primary substances, guidance of themedicine to the proper organs or exertion of a harmonizing effect.

III. Herbs with Antimicrobial Activities Described in the ChinesePharmacopoeia

Many of the 5767 Chinese medicines (herbs, animals and minerals) listedin the Encyclopedia of Traditional Chinese Medicinal Substances (1977)have been used to combat microbial infections.

The herb Zi-Cao (literally translated as “Purple Herb”), is the dry rootof Lithospermum erythrorhizon Sieb, et Zucc. or Arnebia euchroma (Royle)Johnst. These plants belong to the family Boraginaceae. This herb isofficially listed in the Chinese Pharmacopoeia, and has frequently beenused as an anti-inflammatory and anti-pyretic agent in the treatment ofmeasles, eczema and thermal burns (Tang et al, Chinese Drugs of PlantOrigin, pages 613-619, Springer-Verlag (1992)). The aqueous or organicextract of these roots has been used in combination with other herbs inthe form of ointments for topical use, or in the form of aqueousinfusions as an antipyretic to “cool the blood and as an antidote tobody toxins induced by heat excess”.

The roots of plants from the family of Boraginaceae containnaphthoquinone pigments as the main chemical constituents (Tang et al,supra). Shikonin and its derivatives are the main naphthoquinonepigments. Shikonin is a naphthoquinone with an unsaturated side chainand an asymmetric centrum bearing a hydroxy group. A series ofcarboxylic acid esters of shikonin have been identified (Morimoto et al,Tetrahedron Lett., 52:4737-4739 (1965); Morimoto et al, TetrahedronLett., 31:3677-3680 (1966); and Kyogoku et al, Shoyakugagu Zasshi,27:24-30 (1973)). The chemical structures of shikonin and its identifiedderivatives are shown in FIG. 1.

The total organic solvent extract from root of a plant belonging to thefamily Boraginaceae, and some of the individual components of theextract, i.e., shikonin and deoxyshikonin, have been identified ashaving antimicrobial activity (Tanaka et al, Yakugaku Zasshi, 92:525-530(1972); Kyogoku et al, Shoyakugaku Zasshi, 27:31-36 (1973); and Honda etal, J. Natural Products, 51:152-154 (1988)); and anti-inflammatoryactivity (Tanaka et al, J. Natural Products, 9:466-469 (1986)). However,there is no teaching or suggestion in the art that the extracts, orcomponents thereof, are effective against antibiotic-resistant bacteria,much less gram-positive antibiotic-resistant bacteria.

SUMMARY OF THE INVENTION

An object of the present invention is to provide antimicrobial agentswhich can effectively inhibit the growth of bacteria by mechanismsdifferent from those of existing antibiotics.

Still another object of the present invention is to provide compositionswhich are useful for treatment of antibiotic-resistant gram-positivebacterial infections.

Yet another object of the present invention is to provide methods fortreatment of antibiotic-resistant gram-positive bacterial infections.

An additional object of the present invention is to provide methods forobtaining said compositions.

These and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met, in one embodiment by a method for the treatment ofantibiotic-resistant gram-positive bacterial infections comprisingadministering to a subject infected with antibiotic-resistantgram-positive bacteria, a pharmaceutically effective amount of a totalorganic solvent extract from root of Boraginaceae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of the major active ingredientsfound in the root of Arnebia euchroma and Lithospermum erythrorhizon.

FIG. 2 is an HPLC chromatogram of a total organic solvent extract fromArnebia euchroma.

FIG. 3 is an HPLC chromatogram of a total organic solvent extract fromLithospermum erythrorhizon.

FIGS. 4A-4B show growth curves of a wild-type strain of Staphylococcusaureus (ATCC 29213) in the presence and absence of a total organicsolvent extract from Arnebia euchroma. FIG. 4A is plotted as OD vs.time; and FIG. 4B is plotted as CFU vs. time.

FIGS. 5A-5B show growth curves of an antibiotic-resistant strain ofStaphylococcus aureus (ATCC 27695) in the presence and absence of atotal organic solvent extract from Arnebia euchroma. FIG. 5A is plottedas OD vs. time; and FIG. 5B is plotted as CFU vs. time.

DETAIL DESCRIPTION OF THE INVENTION

As discussed above, in one embodiment, the present invention relates toa method for the treatment of antibiotic-resistant gram-positivebacterial infections comprising administering to a subject infected withantibiotic-resistant gram-positive bacteria, a pharmaceuticallyeffective amount of a total organic solvent extract from root ofBoraginaceae.

As used herein “total organic solvent extract” means the total solidmaterial obtained by extracting the dry roots of herbs with an organicsolvent.

The organic solvent used to obtain the extract is not critical to thepresent invention. Examples of such organic solvents include sesame seedoil, methanol, ethanol, chloroform, and ether. Sesame seed oil andmethanol are the preferred organic solvents used to obtain the extracts.

Plants of the Boraginaceae family whose root is employed is not criticalto the present invention. Examples of such plants include Arnebiaeuchroma, Lithospermum erythrorhizon, and Arnebia guttata. Arnebiaeuchroma is the preferred plant used in the present invention.

The ratio of root to solvent used to prepare the extract is generallyabout 1:20 to 1:5 (w/v), preferably about 1:10 (w/v).

It is preferable when preparing the extract to minimize exposure tolight, and high temperatures, e.g., temperatures above 50° C. should beavoided.

The total organic solvent extract of the present invention generallycomprises from about 0.05-0.25% (w/w) shikonin, from about 1.8-2.8%(w/w) acetyl shikonin, from about 0-0.8% (w/w) teracyl shikonin, fromabout 0.15-1.5% (w/w) hydroxyisovaleryl shikonin, from about 2.0-6.5%(w/w) isobutyl shikonin, and from about 1.5-4.5% (w/w) β,β-dimethylacrylshikonin. Preferably, the total organic solvent extract of the presentinvention comprises from about 0.1-0.2% (w/w) shikonin, from about2.0-2.5% (w/w) acetyl shikonin, from about 0.05-0.5% (w/w) teracylshikonin, from about 0.2-1.0% (w/w) hydroxyisovaleryl shikonin, fromabout 2.5-5.5% (w/w) isobutyl shikonin, and from about 2.5-4.0% (w/w)β,β-dimethylacryl shikonin.

In another embodiment, the present invention relates to a method for thetreatment of antibiotic-resistant gram-positive bacterial infectionscomprising administering to a subject infected with antibiotic-resistantgram-positive bacteria, a pharmaceutically effective amount of a redcolored active fraction of a total organic solvent extract from root ofBoraginaceae.

As used herein “red colored active fraction of a total organic solventextract” means the red colored fraction of the total organic solventextract obtained by thin layer chromatography (TLC) or a silica gelcolumn and chloroform as the developing solvent. The major components inthis red colored fraction are naphthoquinones, and thus the fraction canalso be referred to as the “naphthoquinone fraction”.

The red colored active fraction of the present invention generallycomprises from about 0.5-2.5% (w/w) shikonin, from about 18-28% (w/v)acetyl shikonin, from about 0-8.0% (w/w) teracyl shikonin, from about1.5-15% (w/w) hydroxyisovaleryl shikonin, from about 20-65% (w/w)isobutyl shikonin, and from about 15-45% (w/w) β,β-dimethylacrylshikonin. Preferably, the red colored active fraction of the presentinvention comprises from about 1.0-2.0% (w/w) shikonin, from about20-25% (w/w) acetyl shikonin, from about 0.5-5.0% (w/w) teracylshikonin, from about 2.0-10% (w/w) hydroxyisovaleryl shikonin, fromabout 25-55% (w/w) isobutyl shikonin, and from about 25-40% (w/w)β,β-dimethylacryl shikonin.

In still another embodiment, the present invention relates to a methodfor the treatment of antibiotic-resistant gram-positive bacterialinfections comprising administering to a subject infected withantibiotic-resistant gram-positive bacteria, a pharmaceuticallyeffective amount of a composition comprising acetyl shikonin andβ,β-dimethylacryl shikonin.

In the composition of the present invention acetyl shikonin andβ,β-dimethylacryl shikonin are each generally present in an amount offrom about 20-60% (w/w), preferably about 25-40% (w/w). Othernaphthoquinone compounds may also be present.

As defined herein “antibiotic-resistant” gram-positive bacteria arethose whose growth cannot be inhibited by commonly used antibiotics,e.g., those listed in Table 1 above. The particular antibiotic-resistantgram-positive bacteria whose growth are inhibited by the treatment ofthe present invention is not critical thereto. Examples of suchantibiotic-resistant gram-positive bacteria include antibiotic-resistantStaphylococcus aureus, Enterococcus faecalis, and Streptococcuspneumoniae.

The particular mode of administration is not critical to the presentinvention. For example, the total organic solvent extract, red coloredfraction or composition can be administered topically or orally. Topicaluse is the preferred mode of administration.

The particular pharmaceutically effective amount of the total organicsolvent extract employed in the present invention is not criticalthereto. The particular amount to be administered in accordance with thepresent invention varies depending upon the mode of administration, thebacteria to be treated, the severity or extent of infection, whetheradministered alone or in combination with other drugs, and the age,weight and sex of the subject to be treated. Generally, the amount to beadministered topically is in the range of about 0.5 to 20 mg/cm²,preferably about 2.0 to 5.0 mg/cm². Generally, the amount to beadministered orally is in the range of about 1.0 to 75 mg/dose,preferably about 10 to 25 mg/dose.

The particular pharmaceutically effective amount of the red coloredactive fraction employed in the present invention is not critical,thereto. The particular amount to be administered in accordance with thepresent invention varies depending upon the mode of administration, thebacteria to be treated, the severity or extent of infection, whetheradministered alone or in combination with other drugs, and the age,weight and sex of the subject to be treated. Generally, the amount to beadministered topically is in the range of about 0.05 to 2.0 mg/cm²,preferably about 0.2 to 0.5 mg/cm². Generally, the amount to beadministered orally is in the range of about 0.1 to 7.5 mg/dose,preferably about 1.0 to 2.5 mg/dose.

The particular pharmaceutically effective amount of the compositionemployed in the present invention is not critical thereto. Theparticular amount to be administered in accordance with the presentinvention varies depending upon the mode of administration, the bacteriato be treated, the severity or extent of infection, whether administeredalone or in combination with other drugs, and the age, weight and sex ofthe subject to be treated, as well as the concentration of acetylshikonin and β,β-dimethylacryl shikonin in the composition. Generally,the amount to be administered topically is in the range of about 0.03 to1.0 mg/cm², preferably about 0.1 to 0.4 mg/cm². Generally, the amount tobe administered orally is in the range of about 0.05 to 4.0 mg/dose,preferably about 0.5 to 1.5 mg/dose.

The total organic solvent extract or red colored active fraction thereofor composition of the present invention can be used alone, or incombination with other herbs, e.g., borneol, Radix agnelicae sinensis,Flos carthami tinctorii, and Radix glycyrrhizae (Hu, Handbook ofClinical Applications of Chinese Herbal Medicines, Beijing (1993)).

Furthermore, the total organic solvent extract or red colored activefraction thereof or composition of the present invention can be used,e.g., in the form of an ointment, a solution or as a cream. The totalorganic solvent extract or red colored active fraction thereof orcomposition can be admixed with any conventional oil, wax petroleumjelly and other inert excipients to obtain ointments and creams; or withconventional organic solvents (e.g., methanol, ethanol or isopropanol)to obtain solutions.

The following Examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

EXAMPLE 1 Preparation of Total Organic Solvent Extract

Dry roots of each of Arnebia euchroma and Lithospermum erythrorhizonwere soaked overnight at room temperature (22-27° C.) in an organicsolvent, e.g., methanol or ethanol, at a ratio of 1 part dry root:10parts solvent (w/v). The resulting mixture was then filtered through aWhatman #1 filter to remove the solid material. The volume of thefiltrate was reduced to 1/10 of its original volume by evaporation, andacidified by the addition of 1/10 volume of 2.0 N HCl (or any otherinorganic acid). The resulting red precipitate was collected bycentrifugation at 26,000×g, and air dried. The resulting sticky residue,i.e., the “total organic solvent extract”, was used in theanti-bacterial assays described in Example 4 below.

EXAMPLE 2 Partial Fractionation of Total Organic Solvent Extract

An active fraction, i.e., the red colored active fraction, of the totalorganic solvent extracts was obtained using thin layer chromatography(TLC) or a silica gel column, as described in detail below.

A. Thin Layer Chromatography

The total organic solvent extract from A. euchroma, obtained asdescribed in Example 1 above, was dissolved in a minimal amount(approximately 1:10 (w/v)) of chloroform, and then applied as a band atthe bottom of a 20×20 cm silica gel plate (Sigma Chemical Co.). Theplate was then developed in chloroform at room temperature (22-27° C.)until the solvent reached the top of the plate. All of the observed redbands were scraped from the plate and combined. Then, a red coloredactive fraction was obtained by packing the silica mix in a glass columnand eluting it with chloroform.

B. Silica Gel Column

Silica gel was packed into a glass column using chloroform as thesolvent. The total organic solvent extract from A. euchroma, obtained asdescribed in Example 1 above, was dissolved in a minimal amount(approximately 1:10 (w/v)) of chloroform, and then layered onto the topof the column. Then, a red colored active fraction was obtained byslowly eluting it from the column with chloroform.

EXAMPLE 3 Composition of Shikonin and Its Derivatives in the TotalOrganic Solvent Extract

The content of shikonin and its derivatives varies with the plantspecies, and the location where the plant is collected. FIGS. 2 and 3represent the High Performance Liquid Chromatography (HPLC) patterns oftotal organic solvent extracts from representative batches of Arnebiaeuchroma and Lithospermum erythrorhizon, respectively. The HPLC wasperformed by a reverse phase C-18 column using a linear gradient of 18to 90% (v/v) acetonitrile in the presence of 2.0% (v/v) acetic acid.

The average content of total naphthaquinone pigments in the totalorganic solvent extracts obtained in Example 1 above were calculatedfrom the optical density coefficient and found to be 6.0% to 10% (w/w).The average content of total naphthaquinone pigments in the red coloredactive fraction obtained in Example 2 above were also calculated fromthe optical density coefficient and found to be >95% (w/w). Thepercentage of each component in a typical batch of the red coloredactive fraction was calculated by comparing with standards of knownweight. The results are shown in Table 2 below.

TABLE 2 Composition of Shikonin and Its Derivatives in the Red ColoredActive Fraction % (w/w) in % (w/w) in Peak A. euchroma L. erythrorhizonI. Shikonin 2.0  0.8 II. Acetyl shikonin 24.0 20.1 III. Teracrylshikonin 4.5 trace IV. Hydroxyisovaleryl 9.0  2.9 shikonin V. Isobutylshikonin 26.0 55.9 VI. β, β-dimethylacryl 34.5 20.3 shikonin

EXAMPLE 4 Antimicrobial Activity

The antimicrobial activities of the total organic solvent extract fromA. euchroma, obtained as described in Example 1 above, and the redcolored active fraction from A. euchroma, obtained as described inExample 2 above, were evaluated using a disk sensitivity assay.

More specifically, wild-type Staphylococcus aureus (ATCC 29212) wasgrown in Luria broth (L-broth) at 37° C. to exponential phase, dilutedto 1.0×10⁷ bacteria/ml, and mixed with minimal medium (M63, +salt andglucose) with 0.15% (w/v) soft agar. The mixture was poured over aminimal agar plate (minimal medium +0.7% (w/v) agar). Filter paper disks(6.0 mm in diameter) were then impregnated with 20 μl of total organicsolvent extract from A. euchroma (containing 10 mg/ml of naphthaquinonecompounds in dimethyl formamide (DMF)); 20 μl of total organic solventextract from L. erythrorhizon (containing 10 mg/ml of naphthaquinonecompounds in DMF); 20 μl of the red colored active fraction from A.euchroma isolated using TLC (10 mg/ml in DMF); 20 μl of the red coloredactive fraction from A. euchroma isolated using the silica gel column(10 mg/ml in DMF); or 20 μl of DMF as control, and placed on the surfaceof the agar plates. The plates were then incubated at 37° C. for 18 hrs.In this assay, clear zones surrounding the disks indicated that therewas inhibition of bacterial growth by the material diffusing from thefilter paper disks. The results are shown in Table 3 below.

TABLE 3 Antimicrobial Activities of Total Organic Solvent Extracts andRed Colored Active Fractions Against Wild-Type S. aureus Test sampleZone of Inhibition (mm) Total extract of A. euchroma 9.0 Total extractof L. erythrorhizon 8.5 TLC isolated fraction 9.0 Silica gel isolatedfraction 8.5 Control 0.0

As shown in Table 3 above, all of the samples were found to be activeagainst the test organism.

A. Total Organic Solvent Extract

1. Activity on Gram-Positive and Gram-Negative Bacteria

The disk assay described above was next carried out using the totalorganic solvent extract from A. euchroma, obtained as described inExample 1 above, and various gram-positive or gram-negativeantibiotic-sensitive bacteria. The results are shown in Table 4 below.

TABLE 4 Activity of Total Organic Solvent Extract onAntibiotic-Sensitive Bacteria Bacteria strain Gram +/− Inhibitory effectStaphylococcus aureus + Yes Streptococcus pyrogenes + Yes Streptococcusepidermis + Yes Enterococcus faecalis + Yes Protens vulgaris + No E.coli K12 − No E. coli IMP4213* − Yes *a hyper-permeable E. coli strain

As shown in Table 4 above, the total organic solvent extract exhibitedgrowth inhibitory activity against almost all the gram-positivebacteria, but not against gram-negative bacteria, unless the latter wasrendered hyper-permeable prior to the addition of the extract.

2. Activity on Antibiotic-Resistant Bacteria and Clinical Isolates

The disk assay described above was carried out usingantibiotic-resistant Staphylococcus aureus (ATCC 27695), and 20 μl ofthe total organic solvent extract from A. euchroma (containing 10 mg/mlof naphthaquinone compounds in DMF); 20 μl of the total organic solventextract from L. erythrorhizon (containing 10 mg/ml of naphthaquinonecompounds in DMF); or 20 μl of DMF as a control. The results are shownin Table 5 below.

TABLE 5 Antimicrobial Activities of Total Organic Solvent Extracts andRed Colored Active Fractions Against Antibiotic-Resistant S. aureus Zoneof Test sample Inhibition (mm) Total extract from A. euchroma 9.5 Totalextract from L. erythrorhizon 9.0 TLC isolated fraction 9.0 Silica gelisolated fraction 9.0 Control 0.0

As shown in Table 5 above, all of the samples exhibited growthinhibitory activity against the antibiotic-resistant S. aureus.

Next, another disk assay was carried out as described above using thetotal organic solvent extract from A. euchroma, obtained as described inExample 1 above, and strains of antibiotic-resistant Staphylococcusaureus, which were obtained as clinical isolates from a hospital inBeijing. The results are shown in Table 6 below.

TABLE 6 Activity of Total Organic Solvent Extract onAntibiotic-Resistant S. aureus Antibiotic Resistance Zone of inhibitionStrain (MIC* μg/ml) (mm) 6515 Penicillin G (>20) 10.87 1206 Tetracycline(>32) 12.18 3-44 Tetracycline, 12.56 Erythromycin (>32) 5-75Erythromycin (>32) 12.17 5-76 Erythromycin (>32) 12.63 5-77 Erythromycin(>32) 11.35 5-78 Erythromycin (4) 12.05 4-44 Piperacillin (>128) 12.243-16 Methicillin, 12.22 Piperacillin (32) 3-20 Methicillin, 11.68Piperacillin (128) 5-37 Methicillin,  0.00 Piperacillin (>128) 5-38Methicillin, 13.69 Piperacillin (32) 5-39 Methicillin, 12.60Piperacillin (128) 5-47 Methicillin, 13.02 Piperacillin (>128) 4-15Piperacillin (32) 12.67 4-50 Piperacillin (32) 12.42 *MIC: MinimumInhibition Concentration

As shown in Table 6 above, the total organic solvent extract exhibitedgrowth inhibition on 15 out of 16 S. aureus clinical isolates from ahospital in Beijing where almost all S. aureus infections are resistantto antibiotic treatment.

As shown in Tables 3 to 6 above, the total organic solvent extract fromthe Chinese herb, A. euchroma, is effective in inhibiting the growth ofa broad spectrum of gram-positive bacteria, including those resistant toantibiotics.

3. Minimum Inhibitory Concentration (MIC)

Next, the MIC for the total organic solvent extract from A. euchroma,obtained as described in Example 1 above, against wild-typeStaphylococcus aureus and antibiotic-resistant Staphylococcus aureuswere determined in liquid culture using L-broth with different amountsof total organic solvent extract, and compared to the MIC for commonantibiotics.

More specifically, different concentrations of total organic solventextract was added to 1.0 ml of L-broth, and inoculated with 50 μl of anovernight growth of bacteria (about 1.0×10⁸ cells). The resultingsamples were incubated overnight at 37° C. The MIC was then determinedby measuring the minimal concentration of extract needed to inhibit thegrowth of the bacteria. The results are shown in Table 7 below.

TABLE 7 Minimum Inhibitory Concentrations (MIC μg/ml) Extract orWild-Type Antibiotic-Resistant Antibiotic Strain Strain Total organic0.5-1.0 0.5-1.0 solvent extract Tetracycline 1.0-2.0 >20 Erythromycin0.0-1.0 >20 Novobiocin 0.0-2.0 >20

As shown in Table 7 above, the total organic solvent extract from A.euchroma inhibited the growth of wild-type bacteria at a minimumconcentration similar to the MIC for the antibiotics used. The sameextract inhibited the growth of antibiotic-resistant bacteria at thesame MIC as for the wild-type bacteria, while the antibiotic-resistantstrain showed resistance to all three of the antibiotics tested.

4. Growth Inhibition in the Presence or Absence of the Total OrganicSolvent Extract

To determine whether the total organic solvent extract from A. euchromaacts in a bactericidal or bacteriostatic manner, growth curves weredetermined.

More specifically, wild-type S. aureus (ATCC 29213) andantibiotic-resistant S. aureus (ATCC 27659) were each grown toexponential phase in L-broth at 37° C., and 2.0 μg/ml of the totalorganic solvent extract was added thereto. The optical density (OD) at600 nm, and the colony forming units (CFU) were then determined atdifferent time intervals. For the control, only solvent, i.e., DMF, wasadded to the bacteria. The results are shown in FIGS. 4A-4B and FIGS.5A-5B, respectively.

As shown in both FIG. 4A (wild-type S. aureus) and FIG. 5A(antibiotic-resistant S. aureus), 30 min after the addition of the totalorganic solvent extract, the total OD, which measures all of thebacterial particles, i.e., live or dead bacteria, ceased to increase,whereas for the control, the OD continued to increase. Further, as shownin both FIG. 4B (wild-type S. aureus) and FIG. 5B (antibiotic-resistantS. aureus), 30 min after the addition of the total organic solventextract, viable cells, measured by their ability to form colonies,decreased rapidly.

The organic solvent extract killed >99% of the microorganisms tested.However, the minute population of surviving bacteria are sensitive tothe total organic solvent extract when regrown in fresh media. Thus, itis likely that the minute population of bacteria that was initiallyresistant to inhibition by the total organic solvent extract was at ametabolic stage that rendered the bacteria insensitive to the totalorganic solvent extract. Upon re-culturing, these cells re-entered a newgrowth cycle, and again become sensitive to the total organic solventextract.

5. Absence of Resistant-Mutant Bacteria

Using conditions that are similar to those commonly used for productionof antibiotic mutants, attempts were made to obtain mutants resistant tothe total organic solvent extract.

More specifically, the total organic extract from A. euchroma, obtainedas described Example 1 above, was mixed with 0.15% (w/v) of agar, andadded to the top of a M63 plate. Then, 1.0 ml of different strains ofovernight growth of antibiotic-resistant S. aureus were added, andincubated at 37° C. overnight. Under similar conditions, a singleantibiotic will generate antibiotic-resistant mutants. However, attemptsto generate mutants resistant to the total organic solvent extract wereunsuccessful.

These results demonstrate that either the target of the total organicsolvent extract for the microorganism is essential and any mutation inthe target is lethal, or that the total organic solvent extract affectsdifferent targets, making it very difficult for the bacteria to overcomethe multiple hits simultaneously.

B. Red Colored Active Fraction

1. Activity on Gram-Positive and Gram-Negative Bacteria

The disk assay described above was used on the gram-positive andgram-negative bacteria shown in Table 4 above using 20 μl of the redcolored active fraction from A. euchroma isolated using TLC (10 mg/ml inDMF); 20 μl of the red colored active fraction from A. euchroma isolatedusing the silica gel column (10 mg/ml in DMF); or 20 μl of DMF ascontrol.

The anti-microbial activities of the red colored active fractionsparallelled those for the total organic solvent extract. That is, thered colored active fractions were active against gram-positive bacteria,but not against gram-negative bacteria, except when the latter wererendered hyper-permeable by genetic manipulation.

2. Activity on Antibiotic-Resistant Bacterial Isolates

The disk assay described above was carried out on wild-type S. aureusand antibiotic-resistant S. aureus using 20 μl of the red colored activefraction from A. euchroma isolated using TLC (10 mg/ml in DMF); 20 μl ofthe red colored active fraction from A. euchroma isolated using thesilica gel column (10 mg/ml in DMF); or 20 μl of DMF as a control. Theresults are shown in Tables 3 and 5 above.

As shown in Tables 3 and 5 above, the activities of the red coloredactive fractions against both the wild-type S. aureus (Table 3) andantibiotic-resistant S. aureus (Table 5) were found to be similar tothat for the total organic solvent extracts.

3. Minimum Inhibition Concentration

The MIC of the red colored active fraction from A. euchroma weredetermined using S. aureus , and the liquid culture method as describedabove. The results are shown in Table 8 below.

TABLE 8 MIC at the Total Organic Solvent Extract and Red Colored ActiveFractions Extract or Fraction MIC (μg/ml) Total organic solvent extract0.5-1.0 Active Fraction obtained by TLC 1.0-2.0 by Silica gel 1.0-2.0

As shown in Table 8 above, while active, the red colored activefractions were somewhat less potent in inhibiting the growth of bacteriathan the total organic solvent extract.

C. Individual Components of Red Colored Active Fraction

Individual components were isolated from the red colored active fractionby preparative TLC and HPLC.

More specifically, the total organic solvent extract was dissolved inchloroform (1:10 (w/v)) and streaked at the bottom of the TLC plate,which was developed in chloroform. The six individual red colored bandswere scraped from the plate, and the pigments were separated from thesilica gel by eluting with chloroform. Those bands which were too closetogether were separated by HPLC using the same conditions described inExample 3 above.

1. Activity on Wild-type and Antibiotic Resistant Strains of S. aureus

The anti-microbial activities of the 6 individual components weredetermined using the disk sensitivity assay described above.

More specifically, filter paper disks impregnated with 20 μl of eachindividual component (2.0 mg/ml) were placed on top of the agar plate.The plates were developed as described in Example 4 above. The resultsare shown in Table 9 below.

TABLE 9 Inhibitory Activities of Individual Components Zone ofinhibition (mm) Wild-Type Antibiotic-resistant Component Strain StrainPeak I 18 18 Peak II 10 17 Peak III 7.5 7.0 Peak IV 7.0 7.0 Peak V 7.07.0 Peak VI 7.0 7.0

As shown in Table 9 above, each of the 6 individual components inhibitedthe growth of both wild-type S. aureus and antibiotic-resistant S.aureus. However, the sizes of the zones of inhibition were different,indicating different rate of diffusion and different inhibitoryactivities. That is, shikonin (Peak I), gave a large zone of inhibition,but the inhibition was not complete, as shown by an opaque, rather thana clear zone. Other components gave smaller, but clear zones ofinhibition.

2. Minimum Inhibition Concentration

The MIC of the six individual components were determined using wild-typeS. aureus and the liquid culture method as described above. The resultsare shown in Table 10 below.

TABLE 10 MIC of the Total Organic Solvent Extract, Red Colored ActiveFraction and Individual Components Extract or component MIC (μg/ml)Total alcoholic extract 0.5-1.0 Fraction obtained by by TLC 1.0-2.0 bySilica gel 1.0-2.0 Peak I 4.0-5.0 Peak II 0.5-1.0 Peak III 4.0-6.0 PeakIV 4.0-6.0 Peak V 2.0-4.0 Peak VI 0.0-2.0

As shown in Table 10 above, among the individual components, Peak II(acetyl shikonin) and Peak VI (β,β-dimethylacryl shikonin) were the mostactive. These two components are also the most prominent peaks whichmake up a total of 60% of the total naphthoquinone pigments in the redcolored fraction.

3. Single Component vs. Mixture

From the data presented above, it is evident that the total organicsolvent extract is the most active preparation for bactericidalactivity. The red colored active fractions had slightly less activity,but are believed to have better toxicity profiles. Some individualcomponents showed comparable antibacterial activity to the mixture foundin the total organic solvent extract or the red colored activefractions. A mixture of the individual components is believed to providea better therapeutic value, since it makes it almost impossible for thebacteria to develop resistance thereto.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed:
 1. A method for the treatment of antibiotic-resistantgram-positive bacterial infections comprising administering to a subjectinfected with antibiotic-resistant gram-positive bacteria, apharmaceutically effective amount of a total organic solvent extractfrom root of Boraginaceae.
 2. The method of claim 1, wherein saidantibiotic-resistant gram-positive bacteria are selected from the groupconsisting of antibiotic-resistant Staphylococcus aureus,antibiotic-resistant Enterococcus faecalis, and antibiotic-resistantStreptococcus pneumoniae.
 3. The method of claim 1, wherein said extractis administered by a mode selected from the group consisting of topicaladministration and oral administration.
 4. The method of claim 3,wherein said pharmaceutically effective amount is about 0.5 to 20 mg/cm²administered topically, and about 1.0 to 75 mg/dose administered orally.5. The method of claim 4, wherein said pharmaceutically effective amountis about 2.0 to 5.0 mg/cm² administered topically, and about 10 to 25mg/dose administered orally.
 6. The method of claim 1, wherein saidBoraginaceae is a member selected from the group consisting of Arnebiaeuchroma, Lithospermum erythrorhizon and Arnebia guttata.
 7. The methodof claim 1, wherein said total organic solvent extract comprises fromabout 0.05-0.25% (w/w) shikonin, from about 1.8-2.8% (w/w) acetylshikonin, from about 0-0.8% (w/w) teracyl shikonin, from about 0.15-1.5%(w/w) hydroxyisovaleryl shikonin, from about 2.0-6.5% (w/w) isobutylshikonin, and from about 1.5-4.5% (w/w) β,β-dimethylacryl shikonin. 8.The method of claim 7, wherein said total organic solvent extractcomprises from about 0.1-0.2% (w/w) shikonin, 2.0-2.5% (w/w) acetylshikonin, from about 0.05-0.5% (w/w) teracyl shikonin, from about0.2-1.0% (w/w) hydroxyisovaleryl shikonin, from about 2.5-5.5% (w/w)isobutyl shikonin, and from about 2.5-4.0% (w/w) β,β-dimethylacrylshikonin.
 9. A method for the treatment of antibiotic-resistantgram-positive bacterial infections comprising administering to a subjectinfected with antibiotic-resistant gram-positive bacteria, apharmaceutically effective amount of a red colored active fraction of atotal organic solvent extract from root of Boraginaceae.
 10. The methodof claim 9, wherein said antibiotic-resistant gram-positive bacteria areselected from the group consisting of antibiotic-resistantStaphylococcus aureus, antibiotic-resistant Enterococcus faecalis, andantibiotic-resistant Streptococcus pneumoniae.
 11. The method of claim9, wherein said red colored active fraction is administered by a modeselected from the group consisting of topical administration and oraladministration.
 12. The method of claim 11, wherein saidpharmaceutically effective amount is about 0.05 to 2.0 mg/cm²administered topically, and about 0.1 to 7.5 mg/dose administeredorally.
 13. The method of claim 12, wherein said pharmaceuticallyeffective amount is about 0.2 to 0.5 mg/cm² administered topically, andabout 1.0 to 2.5 mg/dose administered orally.
 14. The method of claim 9,wherein said Boraginaceae is a member selected from the group consistingof Arnebia euchroma, Lithospermum erythrorhizon and Arnebia guttata. 15.The method of claim 9, wherein said red colored active fractioncomprises from about 0.5-2.5% (w/w) shikonin, from about 18-28% (w/v)acetyl shikonin, from about 0-8.0% (w/w) teracyl shikonin, from about1.5-15% (w/w) hydroxyisovaleryl shikonin, from about 20-65% (w/w)isobutyl shikonin, and from about 15-45% (w/w) β,β-dimethylacrylshikonin.
 16. The method of claim 15, wherein said red colored activefraction comprises from about 1.0-2.0% (w/w) shikonin, from about 20-25%(w/w) acetyl shikonin, from about 0.5-5.0% (w/w) teracyl shikonin, fromabout 2.0-10% (w/w) hydroxyisovaleryl shikonin, from about 25-55% (w/w)isobutyl shikonin, and from about 25-40% (w/w) β,β-dimethylacrylshikonin.
 17. The method of claim 1, wherein said antibiotic-resistantgram-positive bacteria are resistant to an antibiotic selected from thegroup consisting of streptomycin, tetracycline, erythromycin, andnorfloxacin.
 18. The method of claim 2, wherein saidantibiotic-resistant gram-positive bacteria are resistant to anantibiotic selected from the group consisting of streptomycin,tetracycline, erythromycin, and norfloxacin.
 19. The method of claim 9,wherein said antibiotic-resistant gram-positive bacteria are resistantto an antibiotic-selected from the group consisting of streptomycin,tetracycline, erythromycin, and norfloxacin.
 20. The method of claim 10,wherein said antibiotic-resistant gram-positive bacteria are resistantto an antibiotic selected from the group consisting of streptomycin,tetracycline, erythromycin, and norfloxacin.